Methods for increasing homologous recombination of a nucleic acid sequence

ABSTRACT

The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host&#39;s genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/374,639, filed Apr. 22, 2002, which application is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for increasing homologousrecombination of a nucleic acid sequence in a filamentous fungus. Thepresent invention also relates to isolated nucleic acid sequencesencoding recombination proteins and to nucleic acid constructs, vectors,and fungal host cells comprising such nucleic acid sequences.

2. Description of the Related Art

The process of genetic engineering relies largely upon the ability oforganisms to take up exogenous DNA and integrate it into their genome.Studies in model organisms have demonstrated that the integration stepis a function of cellular DNA repair pathways that normally operate tomaintain genomic integrity in response to DNA damage that occurs bothspontaneously and as a result of exposure to a variety of exogenousagents such as ionizing radiation, ultraviolet light, and chemicalmutagens (see, Nickoloff, J. A., and M. F. Hoekstra, 1998, Double-strandbreak and recombinational repair in Saccharomyces cerevisiae, p.335-362. In J. A. Nickoloff, and M. F. Hoekstra (ed.), DNA Damage andRepair, Vol. I: DNA repair in prokaryotes and lower eukaryotes. HumanaPress, Totowa, N.J.; Paques, F., and J. E. Haber, 1999, Microbiol. Mol.Biol. Rev. 63: 349-404; Shinohara et al., 1998, Genes Cells 3:145-56).

Integration of exogenous DNA occurs primarily through one of two majorrepair pathways, (1) non-homologous end joining or (2) homologousrecombination. Non-homologous end joining is the direct rejoining ofbroken DNA ends that share little or no homology. The ends frequentlyrequire nuclease-processing before they can be ligated together, andthus non-homologous end joining is often error-prone. In contrast,homologous recombination utilizes an undamaged DNA molecule as atemplate to repair DNA damage in another molecule that shares homologywith the undamaged one. This process is more likely to be error-freethan non-homologous end joining. Techniques in genetic engineering suchas gene replacement or disruption and site-specific integration relyupon homologous recombination. By manipulating the relative contributionof homologous recombination versus non-homologous end joining to overallgenome repair, it should be possible to gain additional control overwhether integration of exogenous DNA occurs in regions of homologyversus more randomly.

In the yeast Saccharomyces cerevisiae, the RAD52 epistasis groupincludes genes that function in meiotic and mitotic homologousrecombination (Nickoloff and Hoekstra, 1998, In J. A. Nickoloff, and M.F. Hoekstra (ed.), DNA Damage and Repair, Vol. I: DNA repair inprokaryotes and lower eukaryotes, p. 335-362, Humana Press, Totowa,N.J.; Osman and Subramani, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58:263-99; Paques and Haber, 1999, supra). Two critical genes in thehomologous recombination pathway are RAD51 and RAD52. The Rad51 proteinforms a stoichiometric nucleoprotein complex and, as judged by in vitroassays, mediates DNA pairing and full, stable strand exchange betweensingle-stranded DNA and homologous duplex DNA (Bianco et al., 1998,Front Bioscience 3: d570-603). The Rad52 protein binds preferentially tosingle-stranded DNA, particularly at ends, and promotes annealingbetween complementary single strands (Mortensen et al., 1996, Proc.Natl. Acad. Sci. USA 93: 10729-10734). In Saccharomyces cerevisiae,RAD52 is required for all known forms of both spontaneous and inducedmitotic homologous recombination. For example, intrachromosomal invertedrepeat recombination is reduced 3000-fold in rad52 (Rattray andSymington, 1994, Genetics 138: 587-595), and plasmid gap repair byhomologous recombination is essentially eliminated (Bartsch et al.,2000, Mol. Cell. Biol. 20: 1194-1205).

There is a need in the art for identifying and isolating recombinationprotein encoded genes from filamentous fungi for use in promotinginterplasmid, plasmid-chromosomal, intrachromosomal, andinterchromosomal homologous recombination.

It is an object of the present invention to provide improved methods forincreasing the homologous recombination of a nucleic acid sequenceintroduced into filamentous fungal cells.

SUMMARY OF THE INVENTION

The present invention relates to methods for increasing the homologousrecombination of a nucleic acid sequence introduced into a filamentousfungal host cell, comprising: (a) introducing into a population offilamentous fungal host cells a first nucleic acid sequence encoding arecombination protein and a second nucleic acid sequence comprising oneor more regions which are homologous with the genome of the filamentousfungal host cell, wherein (i) the recombination protein promotes therecombination of the one or more regions with the correspondinghomologous region in the genome of the filamentous fungal host cell toincorporate the second nucleic acid sequence therein by homologousrecombination, and (ii) the number of host cells comprising theincorporated second nucleic acid sequence in the population of thefilamentous fungal host cells is increased at least 20% compared to thesame population of filamentous fungal host cells without the firstnucleic acid sequence; (b) and isolating from the population of thefilamentous fungal host cells a filamentous fungal cell comprising theincorporated second nucleic acid sequence.

The present invention also relates to methods for producing apolypeptide in a filamentous fungal cell, comprising: (A) cultivatingthe filamentous fungal cell in a medium suitable for production of thepolypeptide, wherein the filamentous fungal cell was obtained by (a)introducing into a population of filamentous fungal host cells a firstnucleic acid sequence encoding a recombination protein and a secondnucleic acid sequence comprising one or more regions which arehomologous with the genome of the filamentous fungal host cell, wherein(i) the recombination protein promotes the recombination of the one ormore regions with the corresponding homologous region in the genome ofthe filamentous fungal host cell to incorporate the second nucleic acidsequence therein by homologous recombination, and (ii) the number ofhost cells comprising the incorporated second nucleic acid sequence inthe population of the filamentous fungal host cells is increased atleast 20% compared to the same population of filamentous fungal hostcells without the first nucleic acid sequence; and (b) isolating fromthe population of filamentous fungal host cells a filamentous fungalcell comprising the incorporated first nucleic acid sequence; and (B)recovering the polypeptide from the cultivation medium.

The present invention also relates to methods for deleting or disruptinga gene in a filamentous fungal cell, comprising: (a) introducing into apopulation of filamentous fungal host cells a first nucleic acidsequence encoding a recombination protein and a second nucleic acidsequence comprising one or more regions which are homologous with thegene of the filamentous fungal host cell, wherein (i) the recombinationprotein promotes the recombination of the one or more regions with thecorresponding homologous region in the genome of the filamentous fungalhost cell to incorporate the second nucleic acid sequence therein byhomologous recombination to delete or disrupt the gene in thefilamentous fungal cell, and (ii) the number of host cells comprisingthe incorporated second nucleic acid sequence in the population of thefilamentous fungal host cells is increased at least 20% compared to thesame population of filamentous fungal host cells without the firstnucleic acid; and (b) isolating from the population of filamentousfungal cells a filamentous fungal cell comprising the deleted ordisrupted gene.

The present invention also relates to nucleic acid sequences encoding arecombination protein selected from the group consisting of: (a) anucleic acid sequence encoding SEQ ID NO:2, or having at least 70%identity with SEQ ID NO:4 or SEQ ID NO:6; (b) a nucleic acid sequencecomprising SEQ ID NO:1, or having at least 70% homology with SEQ ID NO:3or SEQ ID NO:5; (c) a nucleic acid sequence which hybridizes undermedium stringency conditions with (i) SEQ ID NO:3 or SEQ ID NO:5, (ii)the cDNA sequence contained in SEQ ID NO:3 or SEQ ID NO:5, or (iii) acomplementary strand of (i) or (ii); (d) an allelic variant of (a), (b),or (c); and (e) a subsequence of (a), (b), (c), or (d), wherein thesubsequence encodes a polypeptide fragment which has recombinationactivity.

The present invention further relates to isolated recombination proteinsencoded by such nucleic acid sequences and to nucleic acid constructs,vectors, and fungal host cells comprising the nucleic acid sequencesencoding recombination proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B show the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus oryzae rdhA gene and encoded recombinationprotein (SEQ ID NOS:1 and 2, respectively).

FIGS. 2A, B, and C show the genomic DNA sequence and the deduced aminoacid sequence of an Aspergillus oryzae rdhB gene and encodedrecombination protein (SEQ ID NOS:3 and 4, respectively).

FIGS. 3A, B, and C show the genomic DNA sequence and the deduced aminoacid sequence of an Aspergillus oryzae rdhD gene and encodedrecombination protein (SEQ ID NOS:5 and 6, respectively).

FIG. 4 shows a restriction map of pPaHa3B.

FIG. 5 shows a restriction map of pSMO145.

FIG. 6 shows a restriction map of pToC202.

FIG. 7 shows a restriction map of pSMO146.

FIG. 8 shows a restriction map of pPH5.

FIG. 9 shows a restriction map of pPH7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for increasing the homologousrecombination of a nucleic acid sequence introduced into a filamentousfungal host cell, comprising: (a) introducing into a population offilamentous fungal host cells a first nucleic acid sequence encoding arecombination protein and a second nucleic acid sequence comprising oneor more regions which are homologous with the genome of the filamentousfungal host cell, wherein (i) the recombination protein promotes therecombination of the one or more regions with the correspondinghomologous region in the genome of the filamentous fungal host cell toincorporate the second nucleic acid sequence therein by homologousrecombination, and (ii) the number of host cells comprising theincorporated second nucleic acid sequence in the population of thefilamentous fungal host cells is increased at least 20% compared to thesame population of filamentous fungal host cells without the firstnucleic acid sequence; (b) and isolating from the population of thefilamentous fungal host cells a filamentous fungal cell comprising theincorporated second nucleic acid sequence.

The methods of the present invention can advantageously elevate levelsof homologous recombination by more than an order of magnitude,particularly by overexpressing genes encoding recombination proteins.For example, genetic engineering in Aspergillus oryzae and many otherfilamentous fungi is impeded by their asexuality and the difficulty increating gene disruptions and other targeted integrations. The presentmethods overcome this difficulty.

In the methods of the present invention, the first nucleic acid sequenceencoding the recombination protein may be any isolated nucleic acidsequence encoding a recombination protein.

The term “recombination protein” is defined herein as a protein thatparticipates in the process of homologous recombination. Representativeexamples from Saccharomyces cerevisiae are Mre11, Rad50, Xrs2, RPA,Rad51, Rad52, Rad54, Rad55, Rad57, and Rad59.

The term“homologous recombination” is defined herein as the processwherein nucleic acids associate with each other in regions of homology,leading to recombination between those sequences. For purposes of thepresent invention, homologous recombination is determined according tothe procedures summarized by Paques and Haber, 1999, Microbiology andMolecular Biology Reviews 63: 349-404.

The term“isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

The term“genome” will be understood to encompass the chromosome(s) andall extrachromosomal elements, e.g., plasmids such as autonomouslyreplicating plasmids of a cell.

In a first embodiment, the present invention relates to isolated nucleicacid sequences encoding recombination proteins having an amino acidsequence which have a degree of identity to SEQ ID NO:2, SEQ ID NO:4 orSEQ ID NO:6 of at least about 70%, preferably at least about 75%,preferably at least about 80%, more preferably at least about 85%, evenmore preferably at least about 90%, most preferably at least about 95%,and even most preferably at least about 97% (hereinafter “homologouspolypeptides”). In a preferred embodiment, the homologous polypeptideshave an amino acid sequence which differs by five amino acids,preferably by four amino acids, more preferably by three amino acids,even more preferably by two amino acids, and most preferably by oneamino acid from SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. For purposes ofthe present invention, the degree of identity between two amino acidsequences is determined by the Clustal method (Higgins, 1989, CABIOS 5:151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=1, gap penalty=3, windows=5,and diagonals=5.

Preferably, the nucleic acid sequences encoding recombination proteinscomprise the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6; or an allelic variant thereof; or a fragment thereof that hasrecombination activity. In a more preferred embodiment, the nucleic acidsequence encoding a recombination protein comprises the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In anotherpreferred embodiment, the nucleic acid sequence encoding a recombinationprotein consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4or SEQ ID NO:6; or an allelic variant thereof; or a fragment thereof,wherein the recombination protein fragment has recombination activity.

The present invention also encompasses nucleic acid sequences whichencode a recombination protein having the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6, which differ from SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5, respectively, by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5 which encode fragments of SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6, which have recombination activity.

A subsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 is a nucleicacid sequence encompassed by SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5except that one or more nucleotides from the 5′ and/or 3′ end have beendeleted. Preferably, a subsequence of SEQ ID NO:1 contains at least 900nucleotides, more preferably at least 945 nucleotides, and mostpreferably at least 990 nucleotides. Preferably, a subsequence of SEQ IDNO:3 contains at least 1500 nucleotides, more preferably at least 1560nucleotides, and most preferably at least 1620 nucleotides. Preferably,a subsequence of SEQ ID NO:5 contains at least 2160 nucleotides, morepreferably at least 2250 nucleotides, and most preferably at least 2350nucleotides.

A fragment of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 is a proteinhaving one or more amino acids deleted from the amino and/or carboxyterminus of this amino acid sequence. Preferably, a fragment of SEQ IDNO:2 contains at least 300 amino acid residues, more preferably at least315 amino acid residues, and most preferably at least 330 amino acidresidues. Preferably, a fragment of SEQ ID NO:4 contains at least 500amino acid residues, more preferably at least 520 amino acid residues,and most preferably at least 540 amino acid residues. Preferably, afragment of SEQ ID NO:6 contains at least 720 amino acid residues, morepreferably at least 750 amino acid residues, and most preferably atleast 780 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedrecombination protein) or may encode recombination proteins havingaltered amino acid sequences. The allelic variant of a recombinationprotein is a recombination protein encoded by an allelic variant of agene.

In a second embodiment, the present invention relates to isolatednucleic acid sequences which have a degree of homology to therecombination protein coding sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5 of at least about 70%, preferably about 75%, preferably about80%, more preferably about 85%, even more preferably about 90%, mostpreferably about 95%, and even most preferably about 97% homology, whichencode an active recombination protein; or allelic variants andsubsequences of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 which encoderecombination protein fragments which have recombination activity. Forpurposes of the present invention, the degree of homology between twonucleic acid sequences is determined by the Wilbur-Lipman method (Wilburand Lipman, 1983, Proceedings of the National Academy of Science USA 80:726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

In a third embodiment, the present invention relates to isolated nucleicacid sequences encoding recombination proteins having recombinationactivity which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with a nucleic acid probe which hybridizes underthe same conditions with (i) SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5,(ii) the cDNA sequence contained in SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5, (iii) a subsequence of (i) or (ii), or a complementary strand of(i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.). The subsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 may beat least 100 contiguous nucleotides or preferably at least 200contiguous nucleotides. Moreover, the subsequence may encode arecombination protein fragment, which has recombination activity.

The nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 ora subsequence thereof, as well as the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6, or a fragment thereof, may be used todesign a nucleic acid probe to identify and clone DNA encodingrecombination proteins having recombination activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA, which hybridizes with the probes describedabove and which encodes a recombination protein having recombinationactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; or asubsequence thereof, the carrier material is used in a Southern blot.For purposes of the present invention, hybridization indicates that thenucleic acid sequence hybridizes to a labeled nucleic acid probecorresponding to the nucleic acid sequence shown in SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5, its complementary strand, or a subsequence thereof,under very low to very high stringency conditions. Molecules to whichthe nucleic acid probe hybridizes under these conditions are detectedusing X-ray film.

In a preferred embodiment, the nucleic acid probe is a nucleic acidsequence which encodes the recombination protein of SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:6; or a subsequence thereof. In another preferredembodiment, the nucleic acid probe is SEQ ID NO:1, SEQ ID NO:3 or SEQ IDNO:5. In another preferred embodiment, the probe is the nucleic acidsequence encoding a recombination protein contained in plasmidpZL1rdhA13 that is contained in Escherichia coli NRRL B-30503. Inanother preferred embodiment, the probe is the nucleic acid sequenceencoding the recombination protein contained in plasmid pZL1rdhB6 thatis contained in Escherichia coli NRRL B-30503. In another preferredembodiment, the probe is the nucleic acid sequence encoding arecombination protein contained in plasmid pZL1rdhD17 that is containedin Escherichia coli NRRL B-30505. In another preferred embodiment, theprobe is the nucleic acid sequence encoding a recombination proteincontained in plasmid pZL1rdhD10 that is contained in Escherichia coliNRRL B-30506.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at 5° C. to 10° C. belowthe calculated T_(m) using the calculation according to Bolton andMcCarthy (1962, Proceedings of the National Academy of Sciences USA48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40,1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasicphosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standardSouthern blotting procedures.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5; (ii) the cDNA sequence contained innucleotides SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5; (iii) a subsequenceof (i) or (ii); or (iv) a complementary strand of (i), (ii), or (iii);and (b) isolating the nucleic acid sequence. The subsequence ispreferably a sequence of at least 100 contiguous nucleotides such as asequence, which encodes a recombination protein fragment which hasrecombination activity.

In a fourth embodiment, the present invention relates to isolatednucleic acid sequences which encode variants of the recombinationprotein having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6, comprising a substitution, deletion, and/or insertion of one ormore amino acids.

The amino acid sequences of the variant recombination proteins maydiffer from the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQID NO:6, by an insertion or deletion of one or more amino acid residuesand/or the substitution of one or more amino acid residues by differentamino acid residues. Preferably, amino acid changes are of a minornature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

Modification of a nucleic acid sequence of the present invention may benecessary for the synthesis of recombination proteins substantiallysimilar to the recombination protein. The term “substantially similar”to the recombination protein refers to non-naturally occurring forms ofthe recombination protein. These recombination proteins may differ insome engineered way from the recombination protein isolated from itsnative source, e.g., variants that differ in specific activity,thermostability, pH optimum, or the like. The variant sequence may beconstructed on the basis of the nucleic acid sequence presented as therecombination protein encoding part of SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the recombination protein encoded by the nucleic acidsequence, but which corresponds to the codon usage of the host organismintended for production of the enzyme, or by introduction of nucleotidesubstitutions which may give rise to a different amino acid sequence.For a general description of nucleotide substitution, see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active recombination protein. Amino acid residuesessential to the activity of the recombination protein encoded by theisolated nucleic acid sequence of the invention, and thereforepreferably not subject to substitution, may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, mutations areintroduced at every positively charged residue in the molecule, and theresultant mutant molecules are tested for recombination activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904;Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The nucleic acid sequences encoding recombination proteins may beobtained from microorganisms of any genus. For purposes of the presentinvention, the term “obtained from” as used herein in connection with agiven source shall mean that the recombination protein encoded by thenucleic acid sequence is produced by the source or by a cell in whichthe nucleic acid sequence from the source has been inserted.

The nucleic acid sequences encoding recombination proteins may beobtained from any filamentous fungal source including, but not limitedto, an Acremonium, Aspergillus, Aureobasidium, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, orTrichoderma strain.

In a preferred embodiment, the nucleic acid sequences are obtained froma Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride strain.

In another preferred embodiment, the nucleic acid sequences are obtainedfrom an Aspergillus aculeatus, Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae strain.

In a more preferred embodiment, the nucleic acid sequences are obtainedfrom Aspergillus oryzae.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such nucleic acid sequences may be identified and obtainedfrom other sources including microorganisms isolated from nature (e.g.,soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms from natural habitats are wellknown in the art. The nucleic acid sequence may then be derived bysimilarly screening a genomic or cDNA library of another microorganism.Once a nucleic acid sequence encoding a polypeptide has been detectedwith the probe(s), the sequence may be isolated or cloned by utilizingtechniques which are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

In a most preferred embodiment, the nucleic acid sequence encoding therecombination protein is set forth in SEQ ID NO:1. In another mostpreferred embodiment, the nucleic acid sequence is the sequencecontained in plasmid pZL1rdhA13 that is contained in Escherichia coliNRRL B-30503. In another most preferred embodiment, the nucleic acidsequence is set forth in SEQ ID NO:3. In another most preferredpreferred embodiment, the nucleic acid sequence encoding therecombination protein is the sequence contained in plasmid pZL1rdhB6that is contained in Escherichia coli NRRL B-30503. In another mostpreferred embodiment, the nucleic acid sequence encoding therecombination protein is set forth in SEQ ID NO:5. In another mostpreferred preferred embodiment, the nucleic acid sequence is thesequence contained in plasmid pZL1rdhD17 that is contained inEscherichia coli NRRL B-30505. In another most preferred embodiment, thenucleic acid sequence encoding the recombination protein is set forth inSEQ ID NO:7. In another most preferred embodiment, the nucleic acidsequence is the sequence contained in plasmid pZL1rdhD10 that iscontained in Escherichia coli NRRL B-30506.

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in the recombination protein codingsequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 in which the mutantnucleic acid sequence encodes a polypeptide which consists of SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:6, respectively.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well-known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Aspergillus, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

In the methods of the present invention, the first nucleic acidsequences encoding recombination proteins are preferably overexpressed.Overexpression of these genes can be accomplished by multiple insertionsof the genes in the genome of the filamentous fungal host cell and/or bysubstituting heterologous control sequences for the native controlsequences in the gene, e.g., a strong promoter.

In the methods of the present invention, the second nucleic acid may beany nucleic acid sequence. The second nucleic acid sequence preferablycomprises (a) a gene that encodes a polypeptide or an RNA; (b) a genedisrupted with a third nucleic acid sequence; (c) a partially or fullydeleted gene; (d) a regulatory control sequence; or (e) a recombinantlymanipulated version of a gene native or foreign to the filamentousfungal host cell.

In a preferred embodiment, the second nucleic acid sequence comprises agene encoding a polypeptide or an RNA. The polypeptide or RNA encoded bythe nucleic acid sequence may be native or heterologous to the fungalhost cell of interest.

The term “polypeptide” is not meant herein to refer to a specific lengthof the encoded product and, therefore, encompasses peptides,oligopeptides, and proteins. The term “heterologous polypeptide” isdefined herein as a polypeptide which is not native to the fungal cell,a native polypeptide in which modifications have been made to alter thenative sequence, or a native polypeptide whose expression isquantitatively altered as a result of a manipulation of the fungal cellby recombinant DNA techniques. For example, a native polypeptide may berecombinantly produced by, e.g., placing a gene encoding the polypeptideunder the control of a promoter sequence. The filamentous fungal cellmay contain one or more copies of the nucleic acid sequence encoding thepolypeptide.

Preferably, the polypeptide is an antibody, hormone, enzyme, receptor,reporter, or selectable marker. In a preferred embodiment, thepolypeptide is secreted extracellularly. In a more preferred embodiment,the polypeptide is an oxidoreductase, transferase, hydrolase, lyase,isomerase, or ligase. In an even more preferred embodiment, thepolypeptide is an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolyticenzyme, peroxidase, phospholipase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The nucleic acid sequence encoding a polypeptide of interest may beobtained from any prokaryotic, eukaryotic, or other source. Thetechniques used to isolate or clone a nucleic acid sequence encoding apolypeptide of interest are known in the art and include isolation fromgenomic DNA, preparation from cDNA, or a combination thereof, asdescribed above. The nucleic acid sequence may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

In the methods of the present invention, the polypeptide may alsoinclude a fused or hybrid polypeptide in which another polypeptide isfused at the N-terminus or the C-terminus of the polypeptide or fragmentthereof. A fused polypeptide is produced by fusing a nucleic acidsequence (or a portion thereof) encoding one polypeptide to a nucleicacid sequence (or a portion thereof) encoding another polypeptide.Techniques for producing fusion polypeptides are known in the art, andinclude, ligating the coding sequences encoding the polypeptides so thatthey are in frame and expression of the fused polypeptide is undercontrol of the same promoter(s) and terminator. The hybrid polypeptidemay comprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or moremay be heterologous to the mutant fungal cell.

The selectable marker gene may be, but is not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase); and equivalents thereof.

In another preferred embodiment, the second nucleic acid sequencecomprises a disrupted gene. The gene may be disrupted with any nucleicacid sequence. In a preferred embodiment, the gene is disrupted with aselectable marker gene selected from the group described above.

In another preferred embodiment, the second nucleic acid sequencecomprises a partially or fully deleted gene. Where the nucleic acidsequence comprises a fully deleted gene, it will be understood that thenucleic acid sequence will contain regions upstream and downstream ofthe gene that are homologous with corresponding homologous regions inthe genome of the filamentous fungal cell.

The second nucleic acid sequence comprising a disrupted or deleted genemay be constructed by using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. The gene to bedisrupted or deleted may be, for example, the coding region or a partthereof essential for activity, or the gene may contain a regulatoryelement required for expression of the coding region. An example of sucha regulatory or control sequence may be a promoter sequence or afunctional part thereof, i.e., a part which is sufficient for affectingexpression of the nucleic acid sequence. Other control sequences forpossible modification include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, signal sequence,transcription terminator, and transcriptional activator.

Disruption or deletion of the gene may be also accomplished byintroduction, substitution, or removal of one or more nucleotides in thegene or a regulatory element required for the transcription ortranslation thereof. For example, nucleotides may be inserted or removedso as to result in the introduction of a stop codon, the removal of thestart codon, or a change of the open reading frame.

An example of a convenient way to disrupt or delete a gene is based ontechniques of gene replacement, gene deletion, or gene disruption. Forexample, in the gene disruption method, a nucleic acid sequencecorresponding to the endogenous gene or gene fragment of interest ismutagenized in vitro to produce a defective nucleic acid sequence whichis then transformed into the parent cell to produce a defective gene. Byhomologous recombination, the defective nucleic acid sequence replacesthe endogenous gene or gene fragment. It may be desirable that thedefective gene or gene fragment also encodes a marker, which may be usedfor selection of transformants in which the nucleic acid sequence hasbeen modified or destroyed. The selectable marker gene may be used toachieve the disruption. The defective nucleic acid sequence may be asimple disruption of the endogenous sequence with a selectable markergene. Alternatively, the defective nucleic acid sequence may contain aninsertion or deletion of the endogenous sequence, or a portion thereof,in addition to the disruption with the selectable marker gene.Furthermore, the defective nucleic acid sequence may contain aninsertion or deletion of the endogenous sequence, or a portion thereof,and the selectable marker gene is not involved in the modification butis used as a selectable marker for identifying transformants containingthe defective gene.

In another preferred embodiment, the second nucleic acid sequencecomprises a regulatory control sequence. The regulatory control sequencecan be any control sequence, including, but not limited to, a promoter,signal sequence, leader, polyadenylation sequence, propeptide sequence,consensus translational initiator sequence, signal peptide sequence, andtranscription terminator.

In another preferred embodiment, the second nucleic acid sequencecomprises a recombinantly manipulated version of a gene native orforeign to the filamentous fungal host cell. Further discussion ofconstructing a recombinantly manipulated version of a gene is discussedbelow.

The second nucleic acid sequence comprises one or more regions, whichare homologous with the genome of the filamentous fungal host cell. Therecombination protein promotes the recombination of the one or moreregions with the corresponding homologous region in the genome of thefilamentous fungal host cell to incorporate the second nucleic acidsequence therein by homologous recombination. In the methods of thepresent invention any region that is homologous with the genome of thefilamentous fungal host cell may be used.

In a preferred embodiment, the one or more regions homologous with thegenome of the filamentous fungal cell can be a 5′ region and/or a 3′region that flank (a) a gene that encodes a polypeptide or an RNA; (b) agene disrupted with a third nucleic acid sequence; (c) a partially gene;(d) a regulatory control sequence; or (e) a recombinantly manipulatedversion of a gene native or foreign to the filamentous fungal host cell.

In another preferred embodiment, the one or more regions homologous withthe genome of the filamentous fungal cell can be the 5′ region and/or a3′ region of (a) a gene that encodes a polypeptide or an RNA; (b) a genedisrupted with a third nucleic acid sequence; (c) a partially or fullydeleted gene; (d) a regulatory control sequence; or (e) a recombinantlymanipulated version of a gene native or foreign to the filamentousfungal host cell.

In another preferred embodiment, the one or more regions homologous withthe genome of the filamentous fungal cell can be a part of a gene nativeor foreign to the filamentous fungal host cell.

In the methods of the present invention, when the second nucleic acidsequence comprises one or more contiguous regions that are homologouswith the genome of the filamentous fungal cell, the second nucleic acidsequence may integrate into the genome by homologous recombination via anumber of possible mechanisms, yielding a variety of recombinant nucleicacid structures. These include but are not limited to completeintegration of the second nucleic acid sequence into the genome,replacement of a portion of the genome by a portion of the secondnucleic acid sequence, or reciprocal exchange of a portion of the genomeand a portion of the second nucleic acid sequence. (see, for example,Paques and Haber, 1999, Microbiology and Molecular Biology Reviews 63:349-404).

Nucleic Acid Constructs

The present invention also relates nucleic acid constructs comprisingthe first nucleic acid sequence and/or the second nucleic acid sequenceoperably linked to one or more control sequences which direct theexpression of the coding sequence in a suitable host cell underconditions compatible with the control sequences. Expression will beunderstood to include any step including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains a codingsequence and all the control sequences required for expression of thecoding sequence.

An isolated nucleic acid sequence encoding a polypeptide may be furthermanipulated in a variety of ways to provide for expression of thepolypeptide. Manipulation of the nucleic acid sequence prior to itsinsertion into a vector may be desirable or necessary depending on theexpression vector. The techniques for modifying nucleic acid sequencesutilizing recombinant DNA methods are well known in the art.

In the methods of the present invention, the nucleic acid sequence maycomprise one or more native control sequences or one or more of thenative control sequences may be replaced with one or more controlsequences foreign to the nucleic acid sequence for improving expressionof the coding sequence in a host cell.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, consensus translational initiatorsequence of the present invention, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences includetranscriptional and translational stop signals. The control sequencesmay be provided with linkers for the purpose of introducing specificrestriction sites facilitating ligation of the control sequences withthe coding region of the nucleic acid sequence encoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO96/00787), as well as the NA2-tpi promoter (a hybrid of the promotersfrom the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase); and mutant, truncated,and hybrid promoters thereof.

The control sequence may be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′-terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′-end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′-endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Rhizomucor miehei aspartic proteinase and Myceliophthorathermophila laccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The present invention also relates to nucleic acid constructs foraltering the expression of a gene encoding a polypeptide which isendogenous to a host cell. The constructs may contain the minimal numberof components necessary for altering expression of the endogenous gene.In one embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) an exon, and (c) a splice-donor site. Uponintroduction of the nucleic acid construct into a cell, the constructinserts by homologous recombination into the cellular genome at theendogenous gene site. The targeting sequence directs the integration ofelements (a)-(c) into the endogenous gene such that elements (b)-(c) areoperably linked to the endogenous gene. In another embodiment, thenucleic acid constructs contain (a) a targeting sequence, (b) an exon,(c) a splice-donor site, (d) an intron, and (e) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(e) such that elements (b)-(e) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

In both embodiments, the introduction of these components results inproduction of a new transcription unit in which expression of theendogenous gene is altered. In essence, the new transcription unit is afusion product of the sequences introduced by the targeting constructsand the endogenous gene. In one embodiment in which the endogenous geneis altered, the gene is activated. In this embodiment, homologousrecombination is used to replace, disrupt, or disable the regulatoryregion normally associated with the endogenous gene of a parent cellthrough the insertion of a regulatory sequence which causes the gene tobe expressed at higher levels than evident in the corresponding parentcell. The activated gene can be further amplified by the inclusion of anamplifiable selectable marker gene in the construct using methods wellknown in the art (see, for example, U.S. Pat. No. 5,641,670). In anotherembodiment in which the endogenous gene is altered, expression of thegene is reduced.

The targeting sequence can be within the endogenous gene, immediatelyadjacent to the gene, within an upstream gene, or upstream of and at adistance from the endogenous gene. One or more targeting sequences canbe used. For example, a circular plasmid or DNA fragment preferablyemploys a single targeting sequence, while a linear plasmid or DNAfragment preferably employs two targeting sequences.

The constructs further contain one or more exons of the endogenous gene.An exon is defined as a DNA sequence which is copied into RNA and ispresent in a mature mRNA molecule such that the exon sequence isin-frame with the coding region of the endogenous gene. The exons can,optionally, contain DNA which encodes one or more amino acids and/orpartially encodes an amino acid. Alternatively, the exon contains DNAwhich corresponds to a 5′-non-encoding region. Where the exogenous exonor exons encode one or more amino acids and/or a portion of an aminoacid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

The splice-donor site of the constructs directs the splicing of one exonto another exon. Typically, the first exon lies 5′-of the second exon,and the splice-donor site overlapping and flanking the first exon on its3′ side recognizes a splice-acceptor site flanking the second exon onthe 5′-side of the second exon. A splice-acceptor site, like asplice-donor site, is a sequence which directs the splicing of one exonto another exon. Acting in conjunction with a splice-donor site, thesplicing apparatus uses a splice-acceptor site to effect the removal ofan intron.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising comprising the first nucleic acid sequence and/or the secondnucleic acid sequence, and transcriptional and translational stopsignals. The various nucleic acid and control sequences described abovemay be joined together to produce a recombinant expression vector whichmay include one or more convenient restriction sites to allow forinsertion or substitution of the promoter and/or nucleic acid sequenceencoding the polypeptide at such sites. Alternatively, the nucleic acidsequence may be expressed by inserting the nucleic acid sequence or anucleic acid construct comprising the consensus translational initiatorsequence and/or sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with a consensustranslational initiator sequence of the present invention and one ormore appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Suitable selectable markers for use in a filamentousfungal host cell include, but are not limited to, amdS (acetamidase),argB (ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaA (nitritereuctase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphatedecarboxylase), sC (sulfate adenyltransferase), trpC (anthranilatesynthase), as well as equivalents thereof. Preferred for use in anAspergillus cell are the amdS and pyrG genes of Aspergillus nidulans orAspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in thefilamentous fungal host cell in question. Examples of yeast origins ofreplication are the 2 micron origin of replication, ARS1, ARS4, thecombination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75: 1433).

More than one copy of the first nucleic acid sequence and/or the secondnucleic acid sequence may be inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

In the methods of the present invention, the first and/or second nucleicacid sequence is contained in a plasmid, an autonomously replicatingplasmid, or linear DNA fragment when introduced into the filamentousfungal host cell. The first and second nucleic acid sequences may be onthe same plasmid, autonomously replicating plasmid, or linear DNAfragment, or on different plasmids, an autonomously replicatingplasmids, or linear DNA fragments. The first nucleic acid sequence maybe introduced into the filamentous fungal host cell prior to orsimultaneously with the second nucleic acid sequence.

The first and/or second nucleic acid sequences may be introduced intothe chromosome or into an autonomously replicating plasmid of thefilamentous fungal host cell.

Host Cells

The present invention also relates to recombinant filamentous fungalhost cells, comprising a first nucleic acid sequence encoding arecombination protein, which is advantageously used in increasing thehomologous recombination of a second nucleic acid sequence introducedinto the filamentous fungal host cell. A vector comprising a nucleicacid sequence encoding a recombination protein is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be any filamentous fungal cell useful in the methodsof the present invention. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In a preferred embodiment, the filamentous fungal host cell is anAcremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma cell.

In a more preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In another mostpreferred embodiment, the filamentous fungal host cell is a Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In a most preferred embodiment, the Fusarium venenatum cell is Fusariumvenenatum A3/5, which was originally deposited as Fusarium graminearumATCC 20334 and recently reclassified as Fusarium venenatum by Yoder andChristianson, 1998, Fungal Genetics and Biology 23: 62-80 and O'Donnellet al., 1998, Fungal Genetics and Biology 23: 57-67; as well astaxonomic equivalents of Fusarium venenatum regardless of the speciesname by which they are currently known. In another preferred embodiment,the Fusarium venenatum cell is a morphological mutant of Fusariumvenenatum A3/5 or Fusarium venenatum ATCC 20334, as disclosed in WO97/26330.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.

In most preferred embodiment, the filamentous fungal host cell,comprises a nucleic acid sequence encoding a recombination proteinselected from the group consisting of: (a) a nucleic acid sequencehaving at least 70% identity with SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6; (b) a nucleic acid sequence having at least 70% homology with SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5; (c) a nucleic acid sequence whichhybridizes under medium stringency conditions with (i) SEQ ID NO:1, SEQID NO:3 or SEQ ID NO:5, (ii) the cDNA sequence contained in SEQ ID NO:1,SEQ ID NO:3 or SEQ ID NO:5, or (iii) a complementary strand of (i) or(ii); and (d) a subsequence of (a), (b), or (c), wherein the subsequenceencodes a polypeptide fragment which has recombination activity.

In the methods of the present invention, the number of host cellscomprising the incorporated second nucleic acid sequence in thepopulation of the filamentous fungal host cells is increased at least20%, preferably at least 50%, more preferably at least 100%, even morepreferably at least 500%, most preferably at least 1000%, and even mostpreferably at least 2000% compared to the same population of filamentousfungal host cells without the first nucleic acid sequence.

Methods of Production

The present invention also relates to methods for producing apolypeptide in a filamentous fungal cell, comprising: (A) cultivatingthe filamentous fungal cell in a medium suitable for production of thepolypeptide, wherein the filamentous fungal cell was obtained by (a)introducing into a population of filamentous fungal host cells a firstnucleic acid sequence encoding a recombination protein and a secondnucleic acid sequence comprising one or more regions which arehomologous with the genome of the filamentous fungal host cell, wherein(i) the recombination protein promotes the recombination of the one ormore regions with the corresponding homologous region in the genome ofthe filamentous fungal host cell to incorporate the second nucleic acidsequence therein by homologous recombination, and (ii) the number ofhost cells comprising the incorporated second nucleic acid sequence inthe population of the filamentous fungal host cells is increased atleast 20% compared to the same population of filamentous fungal hostcells without the first nucleic acid sequence; and (b) isolating fromthe population of filamentous fungal host cells a filamentous fungalcell comprising the incorporated first nucleic acid sequence; and (B)recovering the polypeptide from the cultivation medium.

The present invention also relates to methods for producing arecombination protein of the present invention comprising (a)cultivating a host cell under conditions conducive for production of therecombination protein; and (b) recovering the polypeptide.

The present invention further relates to methods for producing apolypeptide comprising (a) cultivating a homologously recombinantfilamentous fungal cell, having incorporated therein a new transcriptionunit comprising a regulatory sequence, an exon, and/or a splice donorsite operably linked to a second exon of an endogenous nucleic acidsequence encoding the polypeptide, under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Themethods are based on the use of gene activation technology, for example,as described in U.S. Pat. No. 5,641,670.

In the above methods of production, the filamentous fungal cellcomprises a nucleic acid sequence encoding a recombination proteinselected from the group consisting of: (a) a nucleic acid sequencehaving at least 70% identity with SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6; (b) a nucleic acid sequence having at least 70% homology with SEQID NO:1, SEQ ID NO:3 or SEQ ID NO:5; (c) a nucleic acid sequence whichhybridizes under medium stringency conditions with (i) SEQ ID NO:1, SEQID NO:3 or SEQ ID NO:5, (ii) the cDNA sequence contained in SEQ ID NO:1,SEQ ID NO:3 or SEQ ID NO:5, or (iii) a complementary strand of (i) or(ii); and (d) a subsequence of (a), (b), or (c), wherein the subsequenceencodes a polypeptide fragment which has recombination activity.

In the production methods of the present invention, the filamentousfungal cells are cultivated in a nutrient medium suitable for productionof the polypeptide using methods known in the art. For example, thecells may be cultivated by shake flask cultivation, small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Aspergillus oryzae HowB101 (A1560, pyrGΔ), Aspergillus oryzae HowB425,Aspergillus oryzae HowB430 (HowB101, lipolase::amdS), Aspergillus oryzaeHowB443 (HowB101, TAKArdhA::basta^(R)), Aspergillus oryzae HowB445(HowB101, TAKArdhB::basta^(R) , Aspergillus oryzae HowB446 (HowB101,niaArdhB::basta^(R)), Aspergillus oryzae SE29-70 (HowB425,hemAΔ5′::pyrG), Aspergillus oryzae PaHa29 (SE29-70, pyrGΔ), Aspergillusoryzae PaHa30 (PaHa29, TAKArdhA::pyrG), Aspergillus oryzae PaHa31(PaHa29, TAKArdhB::pyrG), Aspergillus oryzae PaHa32 (PaHa29,niaArdhA::pyrG), Aspergillus oryzae PaHa33 (PaHa29, niaArdhB::pyrG),Aspergillus oryzae PaHa31-2.2 (PaHa31, hemAΔ3′::amdS), Aspergillusoryzae PaHa32-4.6 (PaHa32, hemAΔ3′::amdS), and Aspergillus oryzaePaHa33-5.1 (PaHa33, hemAΔ3′::amdS).

Example 1 Aspergillus oryzae Genomic DNA Extraction

Aspergillus oryzae HowB101, Aspergillus oryzae HowB430, or Aspergillusoryzae HowB425 was grown in 25 ml of 0.5% yeast extract-2% glucose (YEG)medium for 24 hours at 37° C. and 250 rpm. Mycelia were then collectedby filtration through Miracloth (Calbiochem, La Jolla, Calif.) andwashed once with 25 ml of 10 mM Tris-1 mM EDTA (TE) buffer. Excessbuffer was drained from the mycelia preparation which was subsequentlyfrozen in liquid nitrogen. The frozen mycelia preparation was ground toa fine powder in an electric coffee grinder, and the powder was added toa disposable plastic centrifuge tube containing 20 ml of TE buffer and 5ml of 20% w/v sodium dodecylsulfate (SDS). The mixture was gentlyinverted several times to ensure mixing, and extracted twice with anequal volume of phenol:chloroform:isoamyl alcohol (25:24:1 v/v/v).Sodium acetate (3 M solution) was added to the extracted sample to afinal concentration of 0.3 M followed by 2.5 volumes of ice cold ethanolto precipitate the DNA. The tube was centrifuged at 15,000×g for 30minutes to pellet the DNA. The DNA pellet was allowed to air-dry for 30minutes before resuspension in 0.5 ml of TE buffer. DNase-freeribonuclease A was added to the resuspended DNA pellet to aconcentration of 100 μg per ml and the mixture was then incubated at 37°C. for 30 minutes. Proteinase K (200 μg/ml) was added and the tube wasincubated an additional one hour at 37° C. Finally, the sample wasextracted twice with phenol:chloroform:isoamyl alcohol and the DNAprecipitated with ethanol. The precipitated DNA was washed with 70%ethanol, dried under vacuum, resuspended in TE buffer, and stored at 4°C.

Example 2 PCR Amplification of a Portion of the Aspergillus oryzae rdhAGene

A portion of the Aspergillus oryzae rdhA (rad51 homolog A) gene wasamplified by hemi-nested degenerate PCR. The first amplificationemployed degenerate primers 971514 and 971515, shown below, coding foramino acids DNVAYAR and MFNPDPK. Primer 971514(DNVAYAR):5′-GAYAAYGTIGCITAYGCNMG-3′ (SEQ ID NO:7) Primer 971515(MFNPDPK):5′-TTIGGRTCNGGRTTRAACAT-3′ (SEQ ID NO:8)

The amplification reactions (30 μl) were prepared using Aspergillusoryzae HB101 genomic DNA as template with the following components:PCRbuffer II (Perkin Elmer, Branchburg, N.J.), 0.25 mM dNTPs, 0.8 μg ofAspergillus oryzae HowB101 genomic DNA, 6.4 μM primer 971514, 3.2 μMprimer 971515, and 1.5 units of Taq DNA polymerase (Perkin Elmer,Branchburg, N.J.). Before amplification, the template DNA was denaturedin a boiling water bath for 5 minutes and quick-cooled on ice. Thereaction was initiated by adding Taq DNA polymerase to the otherreaction components at 72° C. The reactions were incubated in aPerkin-Elmer Model 480 Thermal Cycler programmed as follows:35 cycleseach for 20 seconds at 94° C., 30 seconds at 66° C., 60 seconds rampingfrom 66 to 50° C., and 60 seconds at 72° C. (5 minute final extension).The reaction products were isolated on a 1.6% agarose gel using 40 mMTris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a300 bp product band was excised from the gel and purified using aQIAquick Gel Extraction Kit (QIAGEN, Chatsworth, Calif.) according tothe manufacturer's instructions.

One-tenth of the isolated 300 bp product was amplified under the sameconditions described above except that primer 971516, shown below, wasused in place of primer 971515. Primer 971516(NQVVAQV):5′-ACYTGIGCIACNACYTGRTT-3′ (SEQ ID NO:9) The products werefractionated as before and a band at approximately 260 bp was excisedand purified as described for the 300 bp product.

The purified PCR product was subsequently subcloned using the TOPO TACloning kit (Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions and the DNA sequence was determined usingM13 Forward (−20) Primer (Invitrogen, Carlsbad, Calif.). DNA sequenceanalysis of the 260 bp rdhA gene segment showed that the amplified genesegment encoded a portion of the corresponding Aspergillus oryzae rdhAgene.

Example 3 Isolation of a Full-length Aspergillus oryzae rdhA GenomicClones

Genomic DNA libraries were constructed using the bacteriophage cloningvector λZipLox (Life Technologies, Gaithersburg, Md.) with E. coliY1090ZL cells (Life Technologies, Gaithersburg, Md.) as a host forplating and purification of recombinant bacteriophage and E. coliDH10Bzip (Life Technologies, Gaithersburg, Md.) for excision ofindividual pZL1 clones containing the rdhA gene.

Aspergillus oryzae HowB425 genomic DNA was partially digested withTsp509I and size-fractionated on 1% agarose gels. DNA fragmentsmigrating in the size range 3-7 kb were excised and eluted from the gelusing Prep-a-Gene reagents (BioRad Laboratories, Hercules, Calif.). Theeluted DNA fragments were ligated with EcoRI-cleaved anddephosphorylated λZipLox vector arms (Life Technologies, Gaithersburg,Md.), and the ligation mixtures were packaged using commercial packagingextracts (Stratagene, La Jolla, Calif.). The packaged DNA libraries wereplated and amplified in Escherichia coli Y1090ZL cells (LifeTechnologies, Gaithersburg, Md.).

The Aspergillus oryzae HowB425 DNA library was plated on NZCYM agarplates. Plaque lifts (Maniatis et al., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)were performed on approximately 40,000 pfu and the DNA was fixed ontomembranes by heating at 80° C. for two hours. The membranes were soakedfor 30 minutes at 65° C. in a hybridization solution containing 6×SSPEand 7.0% SDS.

The subcloned rdhA product of the PCR amplification described in Example2 was excised from the vector pCR2.1-TOPO by digestion with EcoRI.Approximately 28 ng was random-primer labeled using a StratagenePrime-It II Kit (Stratagene, La Jolla, Calif.) according to themanufacturer's instructions and used to probe the approximately 40,000pfu of the Aspergillus oryzae genomic library constructed fromAspergillus oryzae strain HowB425 in the vector λZipLox. Theradiolabeled rdhA gene fragment was then denatured by adding sodiumhydroxide to a final concentration of 0.5 M, and added to thehybridization solution at an activity of approximately 1×10⁶ cpm per mlof hybridization solution. The mixture was incubated overnight at 65° C.in a shaking water bath. Following incubation, the membranes were washedtwo times in 0.2×SSC with 0.2% SDS at room temperature and an additionaltwo times in the same solution at 65° C. The membranes were thensandwiched between sheets of plastic and exposed to X-ray film for 18hours at −80° C. with intensifying screens (Kodak, Rochester, N.Y.).

Fourteen plaques produced strong hybridization signals with the probe.Twelve of the plaques were picked from the plates and eluted overnightin 1 ml of SM (5.8 g/l NaCl, 2 g/l MgSO₄.7H₂O, 50 mM Tris-CI, 0.01%gelatin). For plaque purification, the eluates were diluted 1:100 and 2μl of the dilution was plated on NZCYM plates together with Y1090ZLplating bacteria. Plaque lifts were prepared and screened as describedabove, and individual plaques were picked into 0.5 ml of SM. The pZL1plasmids were excised from the purified phagemid clones according to theprotocol suggested by Life Technologies (Gaithersburg, Md.). Colonieswere inoculated into three ml of LB plus 50 μg/ml ampicillin medium andgrown overnight at 37° C. Miniprep DNA was prepared from each of theseclones using the Qiagen Bio Robot 9600 according to the manufacturer'sprotocol. The plasmids were digested with EcoRI and XbaI andfractionated by agarose gel electrophoresis in order to determine if theclones were identical and to determine their sizes. The nine uniqueclones had insert sizes ranging from 3.15 to 6.4 kb.

Example 4 Characterization of the Aspergillus oryzae Genomic CloneEncoding RDHA

DNA sequencing of each clone was performed with an Applied BiosystemsPrism 377 DNA Sequencer using the BigDye Terminator Cycle SequencingReady Reaction kit (ABI, Foster City, Calif.) according to themanufacturer's instructions. Oligonucleotide sequencing primers weredesigned to complementary sequences in the pZL1 plasmid vector and weresynthesized by Operon Technologies Inc., Alameda, Calif. Contigsequences were generated by sequencing from the ends of each pZL1 cloneand by sequencing subclones obtained from SalI, PstI, or HindIII digestsof Clone #3, Clone #7, Clone #12, or Clone 13.

The 1.3 kb genomic region encompassing the coding sequence was sequencedto an average redundancy of 5.9. The nucleotide sequence and deducedamino acid sequence are shown in FIG. 1 (SEQ ID NOs:1 and 2). Sequenceanalysis of the cloned insert revealed a coding sequence of 1307 bp(excluding the stop codon) encoding a protein of 348 amino acids. Thecoding sequence is punctuated by three introns of 97 bp, 98 bp, and 68bp. The G+C content of the coding sequence is 55.3%. The predicted RDHApolypeptide has a molecular mass of 37.6 kdaI and an isoelectric pointof 5.24. Using the Signal P software program (Nielsen et al., 1997,Protein Engineering 10:1-6), no signal peptide was predicted (Y<0.027).

A comparative alignment of the Aspergillus oryzae RDHA protein sequencewith other sequences using the Clustal W algorithm in the Megalignprogram of DNASTAR, showed that the deduced amino acid sequence of theAspergillus oryzae RDHA protein shares 98% identity to the deduced aminoacid sequence of the UVSC protein of Emericella nidulans (accessionnumber CAB02454).

Clone 13 was deposited as E. coli pZL1rdhA13 (NRRL B-30503) on Jul. 27,2001, with the Agricultural Research Service Patent Culture Collection,Northern Regional Research Center, 1815 University Street, Peoria, Ill.

Example 5 PCR Amplification of a Portion of the Aspergillus oryzae rdhbGene

A portion of two Aspergillus oryzae genes homologous to the yeast rad52gene were amplified by consensus/degenerate PCR (Rose et al., 1998,Nucleic Acids Res. 26:1628-35). The amplification employed primers980539 and 980540 shown below.

Primer 980539 (ANEVFGFNGW):

-   5′-CGAACGAAGTCTTCGGTTTYAAYGGNTGG-3′ (SEQ ID NO:10)    Primer 980540 (KKEGTTDGMK):-   5′-CTTCATGCCGTCGGTAGTNCCYTCYTTYTT-3′ (SEQ ID NO:11)

The amplification reaction (30 μl) was prepared using Aspergillus oryzaeHB425 genomic DNA as template with the following components:PCR bufferII (Perkin Elmer), 0.20 mM dNTPs, 0.4 μg of Aspergillus oryzae HowB425genomic DNA, 5.0 μM primer 980539, 5.0 μM primer 980540, and 3.0 unitsof Taq DNA polymerase. Before amplification, the template DNA wasdenatured in a boiling water bath for 5 minutes and quick-cooled on ice.The reaction was initiated by adding Taq DNA polymerase to the otherreaction components at 72° C. The reactions were incubated in aStratagene Robocycler programmed for 35 cycles each for 30 seconds at94° C., 60 seconds at 53° C., and 90 seconds at 72° C. (7 minutes finalextension).

The amplification products were fractionated as described above for therdhA gene, and bands at about 350 and 300 bp were excised and clonedusing the TOPO TA cloning kit according to the manufacturer'sinstructions and the DNA sequence was determined using T7 promoterprimer. DNA sequence analysis of the 350 and 300 bp gene segments showedthat the amplified gene segments encoded a portion of two closelyrelated Aspergillus oryzae genes, hereafter designated as rdhB (rad52homolog B) and rdhC (rad52 homolog C), respectively.

Example 6 Isolation of a Full-length Aspergillus oryzae rdhb GenomicClone

Approximately 50 ng of the gel-purified ca. 300-bp product of the PCRamplification described in Example 3 was random-primer labeled using aStratagene Prime-It II Kit according to the manufacturer's instructionsand used to probe approximately 100,000 pfu of an Aspergillus oryzaegenomic library constructed from Aspergillus oryzae strain HowB430 inthe vector λZipLox using the same procedures described in Example 3.

Eleven hybridizing plaques were obtained, and four of these werepurified, excised as pZL1 clones, and characterized as described inExample 3. The two unique clones obtained had insert sizes ofapproximately 3.9 kb and 6.3 kb. The larger clone was designated E. colipZL1 clone #6 and submitted to sequence analysis (see Example 7).

Example 7 Characterization of the Aspergillus oryzae Genomic CloneEncoding RDHB

DNA sequencing of each clone was performed with an Applied BiosystemsPrism 377 DNA Sequencer using the BigDye Terminator Cycle SequencingReady Reaction kit according to the manufacturer's instructions.Oligonucleotide sequencing primers were designed to complementarysequences in the pZL1 plasmid vector and were synthesized by OperonTechnologies Inc., Alameda, Calif. Contig sequences were generated usinga transposon insertion strategy (Primer Island Transposition Kit,Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.).

A 3257 bp genomic fragment was sequenced to an average redundancy of4.7. The nucleotide sequence and deduced amino acid sequence are shownin FIG. 2 (SEQ ID NOs:3 and 4). Sequence analysis of the cloned insertrevealed a coding sequence of 1946 bp (excluding the stop codon)encoding a protein of 565 amino acids. The coding sequence is punctuatedby four introns of 78 bp, 65 bp, 56, and 52 bp. The G+C content of thecoding sequence is 51.8%. The predicted RDHB polypeptide has a molecularmass of 60.7 kdaI and an isoelectric point of 8.64. Using the Signal Psoftware program (Nielsen et al., 1997, Protein Engineering 10:1-6), nosignal peptide was predicted (Y<0.043).

A comparative alignment of the Aspergillus oryzae RDHB protein sequencewith other sequences using the Clustal W algorithm in the Megalignprogram of DNASTAR, showed that the deduced amino acid sequence of theAspergillus oryzae RDHB protein shares 33% identity to the deduced aminoacid sequence of the RAD22 protein of Schizosaccharomyces pombe(accession number P36592) and 33% identity to the RAD52 protein ofSaccharomyces cerevisiae (accession number P06778).

Clone #6 was deposited as E. coli pZL1rdhB6 (NRRL B-30504) on Jul. 27,2001, with the Agricultural Research Service Patent Culture Collection,Northern Regional Research Center, 1815 University Street, Peoria, Ill.

Example 8 Construction of pRamB33

Intermediates pRaMB31 and pRaMB32 were constructed as follows:First,plasmid pUC19 was digested with NdeI plus PvuII and the 2241 bp vectorfragment, purfied by agarose gel electrophoresis, was ligated with thefollowing synthetic linker which contains restriction sites for MunI,PacI, BamHI, HindIII, PmeI, and MunI while inactivating the NdeI cloningsite:

(SEQ ID NO:12) 5′-TATCAATTCTTAATTAAGGATCCAAGCTTGTTTAAACAATTC-3′ (SEQ IDNO:13) 3′-AGTTAACAATTAATTCCTAGGTTCGAACAAATTTGTTAAC-5′-

The resulting pUC19-derivative was termed pRaMB31. Next, the Aspergillusoryzae pgk promoter and terminator regions (Genbank accession numberD28484) as well as the bar gene from Streptomyces hygroscopicus (Whiteet al. 1990, Nucleic Acids Res. 18:1062) were amplified by PCR using thefollowing primer pairs:

Aspergillus oryzae pgk promoter:

-   5′-GATACATGTTATGGAGATGTTCTATCACACAAG-3′ (contains AflIII site) (SEQ    ID NO:14)-   5′-CAGGATCCTGCAGTATTGACTACTATGGT-3′ (contains BamHI site) (SEQ ID    NO:15)    Aspergillus oryzae pgk terminator-   5′-CTGTTTAAACTGCAGGGAGGAACTGAAAAAGG-3′ (contains PmeI site) (SEQ ID    NO:16)-   5′-GTTAAGCTTGCGAAACGCAAATAATGTGTTG-3′ (contains HindIII site) (SEQ    ID NO:17)    Streptomyces hygroscopicus bar gene-   5′-GTTACATGTCTCCAGAACGACGCCCGGCGGACATC-3′ (contains AflIII site)    (SEQ ID NO:18)-   5′-TGAAGCTTCAGATCTCGGTGACGGGCAG-3′ (contains HindIII site) (SEQ ID    NO:19)

The amplification reactions (100 μl) was prepared using pMT1612 (whichharbors the bar gene from Streptomyces hygroscopicus— EMBL accessionnumber X05822) as template with the following components:1× Pwo buffer(Roche Molecular Biochemicals, Indianapolis, Ind.), 0.25 mM dNTPs, 1.0μM of each primer, and 5 units of Pwo DNA polymerase. The reactions wereincubated in an Applied Biosystems thermocycler programmed for 1 cycleat 95° C. for 3 minutes, 45° C. for 2 minutes, and 67° C. for 5 minutesfollowed by 30 cycle at 95° C. for 2 minutes; 45° C. for 2 minutes; and67° C. for 2 minutes.

The PCR-amplified pgk terminator was digested with HindIII plus PmeI andthe 635 bp product was purified by agarose gel electrophoresis, thenligated with pRaMB31 that had been cleaved with the same enzymes. Theresulting intermediate plasmid was designated as pRaMB31.1. Next, thepgk promoter and bar gene segments were digested with BamHI plus AflIIIand HindIII plus AflIII, respectively, and purified by electrophoresis.These two fragments were combined in a three-part ligation with theintermediate pRaMB31.1 that had been digested with BamHI plus HindIII.The product of this ligation, pRaMB32 contained the Streptomyceshygroscopicus bar gene under transcriptional control of the Aspergillusoryzae pgk promoter and terminator regions.

Next, the Aspergillus oryzae niaA promoter and alkaline protease (alp)terminator regions were amplified by PCR using high-fidelity Pwopolymerase (Boehringer-Mannheim, Indianapolis, Ind.) as above with thefollowing primer pairs:

Aspergillus oryzae niaA promoter

-   5′-GGTTAATTAACCGGCAGGGAAGGCCAATGAAAG-3′(contains AflIII site) (SEQ    ID NO:20)-   5′-CCACGCGTATTTAAATGTCCGGGATGGATAGCACTGTGG-3′ (contains PacI site)    (SEQ ID NO:21)    Aspergillus oryzae alp terminator-   5′-GGACGCGTGCGGCCGCGTACCAGGAGTACGTCGCAGG-3′(contains MluI site) (SEQ    ID NO:22)-   5′-GGAGATCTGCAGCTGTGTACCAATAGAC-3′ (contains BglII site) (SEQ ID    NO:23)

The amplified niaA promoter segment was cloned directly into pUC118(Yanisch-Perron et al, 1985, Gene 33:103-119), which had been digestedwith SmaI and dephosphorylated. Similarly, the alp terminator region wassubcloned into pCR-blunt (Invitrogen, Carlsbad, Calif.). The nucleotidesequences of both products were determined to ensure accuracy. The niaApromoter fragment was isolated by gel electrophoresis following cleavagewith PacI plus MluI, and the alp terminator segment was purified afterdigestion with MluI plus BglII. These purified fragments were mixed in athree-part ligation with pRaMB32 which had been previously cut withBamHl plus PacI. The resulting vector, designated as pRaMB33, contained(a) a selectable bar gene under the transcriptional control of the pgkpromoter and terminator, and (b) unique NotI and SwaI restriction siteslocated between the niaA promoter and alp terminator for directionalcloning of cDNA or other coding regions of interest.

Example 9 Construction of Expression Vector with niaA promoter

Plasmid pRaMB33 was digested with XbaI and NruI to remove theBasta-resistance cassette. The remaining vector was isolated on a 0.8%agarose gel using TAE buffer where a 4.4 kb band was excised from thegel and purified using a QIAquick Gel Extraction Kit (QIAGEN,Chatsworth, Calif.) according to the manufacturer's instructions.

Plasmid pBANe13 (WO 97/47746) was digested with PmeI and NheI, and thefragment containing the pyrG gene and AMG terminator was similarly gelisolated and purified. The fragments were mixed together, blunt-endedusing Klenow polymerase, ligated, and transformed into E. coli DH5α.Plasmid DNA was prepared from ten of the resulting transformants, andone displaying the correct restriction digest pattern was designatedpPaHa3B (FIG. 4). The niaA promoter is induced by nitrate.

Example 10 Plasmids for Inter-plasmid Recombination Assay

Plasmid pSMO122 (U.S. Pat. No. 5,958,727) was digested with HindIII andtreated with bacterial alkaline phosphatase. Plasmid Arp1 (Gems et al.,1991, Gene 98:61-67) was digested with HindIII and the digestfractionated on a 1.0% agarose gel in TAE buffer. A 5.8 kb fragment wasexcised from the gel and purified using a QIAquick Gel Extraction Kit(QIAGEN, Chatsworth, Calif.) according to the manufacturer'sinstructions. This fragment was ligated to the linearized pSMO122plasmid and transformed into Escherichia coli DH5α. Plasmid DNA wasprepared from transformants, and one, showing the correct fragment sizesafter digestion with HindIII, was designated pHB217. The fragmentcontains the AMA1 replication region from Emericella nidulans and thepyrG gene from Aspergillus oryzae.

Plasmid pPaHa1-1 was digested NsiI and the ends were made blunt using T4DNA polymerase. The products were fractionated on a 0.8% agarose gelusing TAE buffer and a 2 kb band was excised from the gel and purifiedusing a QIAEX Gel Extraction Kit (QIAGEN, Chatsworth, Calif.) accordingto the manufacturer's instructions. The fragment was then inserted intothe SmaI site of pHB217. The plasmid was designated pSMO145 (FIG. 5).The plasmid carries a 220 bp deletion of the Emericella nidulans amdSgene encompassing a portion of that gene's promoter, all of the5′-untranslated region, and 132 bp of the coding region.

Plasmid pToC202 (FIG. 6) was constructed to contain three up promotermutations have identified within the Aspergillus nidulans amdS gene:TheI666 and I66 up mutations have been described by Katz et al., 1990, Mol.Gen. Genet 220:373-376. The I9 mutation has been described by Davis andHynes, 1989, TIG 5:14-19 and by Todd, 1998, EMBO 17:2042-2054. PlasmidpI66PI9 contains the Aspergillus nidulans amdS with the two up promotermutations I66 and I9. The amdS allele of this plasmid was subcloned intopUC19 as a 2.7 kb XbaI fragment to form the plasmid pToC186C.(Yanisch-Perron et al., 1985, Gene 33 103-119).

Plasmid pMSX-6B1 contains the Aspergillus nidulans amdS gene with the uppromoter mutation I666. The amdS allele of this plasmid was subclonedinto pUC19 as a 2.7 kb XbaI fragment to form the plasmid pToC196. The I9and I666 mutations were combined by inserting a 544 bp XmaI fragmentfrom pToC186 harboring the I9 mutation into the 4903 bp XmaI fragment ofpToC196 to form the plasmid pToC202 (FIG. 6).

A 3′ truncation of the Emericella nidulans amdS gene was produced bydigesting plasmid pToC202 with EcoRI and HpaI, blunting with Klenowfragment, gel and purified using a QIAEX Gel Extraction Kit according tothe manufacturer's instructions. The fragment was then inserted into theSmaI site of pHB217. The resulting plasmid was designated pSMO146 (FIG.7). The promoter region of amdS in this construct contained mutationsthat enhance promoter strength, allowing good growth on acetamide as thesole nitrogen source with a single copy of the gene.

Example 11 Construction of Aspergillus oryzae rdhA and rdhBOverexpression Vectors

Plasmid pRaMB32 (described in Example 8) was digested with PstI and ScaIand fractionated on a 1% agarose gel. The 2.8 kb band containing the pgkpromoter, bar gene, and pgk terminator was excised and purified with theQiagen QIAEX II kit (QIAGEN, Chatsworth, Calif.) according to themanufacturer's instructions. Plasmid pBANe8 (U.S. Pat. No. 5,958,727)was digested with NsiI and dephosphorylated using 150 units of bacterialalkaline phosphatase followed by heat inactivation at 65° C. for 1 hour.The digest was fractionated on a 1% agarose gel and the 5.0 kb band wasexcised and purified as above. The two fragments were ligated togetherand transformed into E. coli XL10 Gold cells (Stratagene, La Jolla,Calif.) according to the manufacturer's instructions. Plasmid DNA wasprepared from transformants and screened for correctness by digestingwith StuI. One plasmid showing the correct digestion pattern was namedpBANe44.

The 1.3 kb coding region of the Aspergillus oryzae rdhA gene wasamplified by PCR from E. coli pZL1 clone #13. Primers incorporated SwaI,PacI, or NotI sites for subsequent cloning and had the followingsequence:

Sense Swa primer (980442):

-   5′-CATTTAAATGATGACGGCGGATATG-3′ (SEQ ID NO:24)    Antisense Pac Primer (980359):-   5′-GTTAATTAATCAGTTGTTTTCCAAGTC-3′ (SEQ ID NO:25)    Antisense Not Primer (980451):-   5′-AGCGGCCGCTCAGTTGTTTTCCAAGTC-3′ (SEQ ID NO:26)

The amplification reaction (50 μl) was composed of the followingcomponents:1× Pwo buffer (Roche Molecular Biochemicals, Indianapolis,Ind.), 0.2 mM dNTPs, 1.0 μM of each primer, 5 units of Pwo DNApolymerase, and approximately 60 ng of heat-denatured clone #13. Thereactions were incubated in a Perkin-Elmer Model 480 Thermal Cyclerprogrammed as follows:22 cycles each at 94° C. for 45 seconds; 55° C.(52° C. for first two cycles) for 45 seconds; 72° C. for 90 seconds, anda final extension at 72° C. for 7 minutes.

The products were fractionated on a 0.8% agarose gel using TAE buffer,and the predominant band at 1.3 kb was excised and purified using theQIAquick Gel Extraction Kit. The products were cloned into pCR®2.1-TOPO(Invitrogen, Carlsbad, Calif.) after addition of 3′ A-overhangsaccording to the manufacturer's suggested protocol.

The 1.3 kb insert from one randomly selected clone was removed bysequential digestion with SwaI and PacI (TAKA promoter construct) orNotI (niaA promoter construct), gel purified, and ligated into similarlydigested pBANe13, pBANe44, or pPaHa3B. The ligation mixtures weretransformed into E. coli DH5α, and clones were screened for the correctinserts by digestion with SwaI and PacI or SwaI and NotI. Miniprep DNAwas sequenced from the ends of both inserts and shown to contain thefull rdhA coding sequence. The constructs were designated pBANe13rad51,pSMO143, and pPaHa3Brad51.

The 1.96 kb coding region of rdhB was amplified essentially as describedabove using pZL1 clone #6 and the following primers:

Sense SwaI Primer (980924):

-   5′-ATTTAAATGATGCCCAACACGACAGACA-3′ (SEQ ID NO:27)    Antisense PacI Primer (980925):-   5′-TTAATTAACTATTGCGGATGTTGTTGCT-3′ (SEQ ID NO:28)    Antisense NotI primer (980826):-   5′-GCGGCCGCCTATTGCGGATGTTGTTGC-3′ (SEQ ID NO:29)

The annealing temperature for the PCR was 60° C. (58° C. for first twocycles). The DNA was subcloned into pCR-Blunt (Invitrogen, Carlsbad,Calif.), and miniprep DNA from clones containing the correct inserts wascloned into pBANe13, pBANe44, pRaMB33, or pPaHa3B as described above.The resulting constructs were named pBANe13rad52, pSMO145, pSMO155 andpPaHa3Brad52, respectively.

Example 12 Construction of Aspergillus oryzae PaHa29

Aspergillus oryzae hemA 5′-deletion strain SE29-70 (Elrod et al., 2000,Current Genetics 38:291-298) was cultured on PDA plates containing5-aminolevulinic acid and uridine to allow for loss of the pyrG gene.Spores from this plate were then plated on minimal plates containingfluoroorotic acid (FOA), uridine, and 5-aminolevulinic acid. EightFOA-resistant colonies were spore purified on minimal plates containing5-aminolevulinic acid and uridine. One of the FOA-resistant colonies wasverified as having a pyrG deletion phenotype by lack of growth onminimal medium containing 5-aminolevulinic acid and by recovery ofprototrophy after transformation of protoplasts (prepared as in Example13) with an autonomously-replicating plasmid carrying the pyrG gene(pHB217). This strain was designated Aspergillus oryzae PaHa29.

Example 13 Construction of Aspergillus oryzae HowB423 and HowB425

Protoplasts of Aspergillus oryzae HowB101 were transformed with pSMO143or pSMO145 and plated on Basta transformation plates.

Protoplasts of Aspergillus oryzae strain HowB101 were prepared accordingto the method of Christensen et al., 1988, Bio/Technology 6:1419-1422.The transformation was conducted with protoplasts at a concentration ofca. 2×10⁷ protoplasts per ml. One hundred μl of protoplasts were placedon ice for 5 minutes with ca. 2 μg of the pSMO143 or pSMO145; 250 μl of60% polyethylene glycol 4000, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂ wasadded, and the protoplasts were incubated at 37° C. for 30 minutes.Three mls of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, and 10 mMCaCl₂) was added. The solution was mixed gently and poured onto 150 mmBasta transformation plates (per liter:0.52 g of KCl, 0.52 g of MgSO₄.7H₂O, 1.52 g of KH₂PO₄, 1 ml of trace metals described below, 342.3 g ofsucrose, 25 g of Noble agar, 10 ml of 1 M urea, 10 ml of 5 mg/ml Basta).The trace metals solution (1000×) was comprised of 22 g of ZnSO₄.7H₂O,11 g of H₃BO₃, 5 g of MnCl₂.4H₂O, 1.6 g of CoCl₂. 5H₂O, 1.6 g of(NH₄)₆Mo₇O₂₄, and 50 g of Na₄EDTA per liter. Plates were incubated 5-7days at 34° C. until colonies appeared. Putative transformants werespore purified twice on the same medium.

Example 14 Construction of hemA 3′-deletion

Plasmid pSE17 (WO 97/47746) was digested with HindIII to remove aportion of the hemA coding sequence and all of the 3′ flanking sequenceto produce a 6.3 kb fragment. The 6.3 kb fragment was run on a 0.8%agarose gel in TAE buffer, excised, and purifed using a QIAEX II GelExtraction Kit (QIAGEN, Chatsworth, Calif.) according to themanufacturer's instructions. The fragment was recircularized by ligationand transformed into E. coli XL1-Blue cells to yield plasmid pPH5 (FIG.8).

The amdS gene from Emericella nidulans was isolated from pToC202 bydigestion with EcoRI, Klenow fill-in, digestion with SphI, and gelpurification as above. The amdS gene fragment was ligated into pPH5digested with SphI and SnaBI and similarly gel purified. The ligationmixture was transformed into E. coli XL1-Blue cells and plasmid DNA wasprepared from twenty-four transformants. One plasmid DNA preparationshowing the correct size fragments upon digestion with SacI, KpnI, orBamHI was designated pPH7 (FIG. 9).

Example 15 Construction of hemAΔ Strains Overexpressing rdhA or rdhB

Protoplasts of Aspergillus oryzae PaHa29 were prepared as described inExample 13 and transformed with several μg of supercoiled pBANe13rad51,pBANe13rad52, pPaHa3Brad51, or pPaHa3Brad52, and plated on minimalmedium containing 30 μg/ml 5-aminolevulinic acid. Individualtransformants were spore purified on MMGAS (per liter:0.5 g of NaCl, 0.5g of MgSO₄. 7H₂0, 2.0 g of KH₂PO₄, 1.2 g of K₂HPO₄, 1 ml of trace metalsdescribed below, 218 g of sorbitol, 20 g of Noble agar, 3.7 g of NH₄Cl,0.1 ml of 1.0 M CaCl₂, and 10 ml of glycerol) plus 5-aminolevulinic acid(pBANe13 transformants) or MMASM (per liter:0.5 g of NaCl, 0.5 g ofMgSO₄.7H₂0, 2.0 g of KH₂PO₄, 1.2 g of K₂HPO₄, 1 ml of trace metalsdescribed below, 20 g of sucrose, 20 g of Noble agar, 3.7 g of NH₄Cl,and 0.1 ml of 1.0 M CaCl₂) plus 5-aminolevulinic acid (pPaHa3Btransformants). The trace metals solution (1000X) was comprised of 10 gof ZnSO₄.7H₂O, 0.4 g of CuSO₄.5H₂O, 0.04 g of Na₂B₄O₇.10H₂O, 0.7 g ofMnSO₄.H₂O, 1.2 g of FeSO₄.7H₂O, 1.6 g of CoCl₂.5H₂O, and 0.8 g ofNa₂MoO₂.2H₂O per liter. Respective transformants from the indicatedplasmids were designated PaHa30, PaHa31, PaHa32, and PaHa33. Multipletransformants of each were generated and are designated by appending anumber, e.g., PaHa31-2.

Example 16 Effect of rdhA or rdhB Overexpression on InterplasmidRecombination

Aspergillus oryzae grows very poorly using acetamide as the solenitrogen source. Growth can be greatly enhanced by introduction of oneor more copies of the amdS gene from Emericella nidulans. Thischaracteristic was used to monitor inter-plasmid recombination byco-transforming Aspergillus oryzae protoplasts with twoautonomously-replicating plasmids, one carrying a deletion in the 5′region of amdS (pSMO145), and the other carrying a deletion in the 3′region (pSMO146). Vigorous growth of transformants on acetamide can onlybe achieved following homologous recombination between the differentplasmids to reconstitute at least one complete amdS gene. Both plasmidsalso carry the pyrG gene in order to assess relative transformationefficiency.

The frequency of recombination in parental (Aspergillus oryzae HowB101)and rdhA (Aspergillus oryzae HowB443) or rdhB (Aspergillus oryzaeHowB445) over-expression strains was assessed by co-transforming withboth plasmids and plating on minimal medium with either nitrate oracetamide as the sole nitrogen sources (Table 1). The sucrose in theseplates partially induces the TAKA promoter. Protoplasts of the indicatedstrains were prepared as described in Example 13 and co-transformed with1.5 μg each of pSMO145 and pSMO146. A portion of the protoplasts wasplated on minimal medium with either nitrate or acetamide as the solenitrogen source, and the number of colonies was counted after six daysof incubation at 37° C. Minimal nitrate plates were composed per literof 6 g of NaNO₃, 0.52 g of KCl, 6.08 g of KH₂PO₄, 0.5 g of MgSO₄.7H₂O,342.3 g of sucrose, 10 g of glucose, 0.004 g of biotin, 20 g of nobleagar, and 1 ml of the trace metals described in Example 15. The mediumwas adjusted to pH 6.5 with NaOH. Minimal acetamide plates (COVE) werecomposed per liter of 10 mM acetamide, 15 mM CsCl, 0.52 g of KCl, 1.52 gof KH₂PO₄, 0.52 g of MgSO₄.7H₂O, 342.3 g of sucrose, 25 g of noble agar,and 1 ml of trace metals. Transformation with either plasmid aloneyielded no transformants on acetamide. Overall transformation efficiencyof the over-expressing strains was somewhat reduced compared to theparental strain, however, inter-plasmid recombination frequencies wereelevated by 14 and 26-fold in the rdhA and rdhB over-expression strains,respectively. In Aspergillus oryzae HowB445, plasmids in almost half ofthe total transformants presumably underwent at least one homologousrecombination event that reconstituted a functional amdS gene.

TABLE 1 Stimulation of interplasmid recombination in rdhA or rdhBoverexpressing strains. HowB101 HowB443 HowB445 Transformants per ng,nitrate 3.43 1.83 1.33 (pyrG selection) Transformats per ng, acetamide0.06 0.46 0.61 (amdS and pyrG selection) Recombination frequency 0.0180.251 0.456 Fold stimulation 1.0 14.4 26.1

Example 17 Effect of rdhA or rdhB Overexpression on InterchromosomalRecombination

The hemA gene of Aspergillus oryzae codes for 5-aminolevulinatesynthase, the first enzyme in heme biosynthesis. Mutants lacking thisenzyme are unable to grow unless the medium is supplemented with5-aminolevulinic acid. The native hemA gene in the rdhB overexpressingAspergillus oryzae strain PaHa31-2 has been replaced by hemA carrying a445-bp deletion in the 5′ region of the coding sequence according to theprocedure described in U.S. Pat. No. 6,100,057, and thus this strainwill not grow on minimal medium. Protoplasts of Aspergillus oryzaePaHa31-2 were transformed with 5 μg of plasmid pPH7 (Example 14) usingthe protocol described in Example 13. This plasmid carries the hemA genewith a deletion of all of the 3′-untranslated region and the last 382 bpof the coding region. The plasmid also contains the E. nidulans amdSgene, and transformants were therefore initially selected on COVE plates(Example 16) containing 20 μg/ml of 5-aminolevulinic acid. One specifictransformant that grew on COVE, but still required 5-aminolevulinic acidfor growth, was spore purified twice and designated Aspergillus oryzaePaHa31-2.2.

Spores from transformant Aspergillus oryzae PaHa31-2.2 were plated onMMGU medium (MMGAS (Example 15) without sorbitol and with 10 mM urea inplace of NH₄Cl) containing increasing concentrations of maltose in orderto induce expression of rdhB in a controlled fashion. Growth on thismedium can only occur if homologous recombination occurs between thesingle-copy chromosomal hemAΔ5′-gene and the chromosomally-integratedplasmid carrying the hemAΔ3′ gene.

The results demonstrated that induction of rdhB expression greatlyincreased the frequency of homologous recombination. Concentrations ofmaltose as low as 0.02% had an obvious stimulatory effect. Most of thecolonies were very slow to first appear and also grew very slowly, evenwhen transferred to new plates not containing maltose. However, thesecolonies grew fairly normally when the medium was supplemented with5-aminolevulinic acid, indicating that the complementation for hemAdeficiency was only partial. Most likely this resulted from a geneconversion event that restored the coding region of hemA in one of thehemA3′ gene copies, but failed to restore the 3′-untranslated region.This could result in relatively low-level expression and incompletecomplementation.

The low concentrations of maltose required to achieve marked stimulationof hemA⁺ colony formation suggested that relatively mild induction ofrdhB transcription was sufficient to maximally promote homologousrecombination. Also, transcription from the TAKA promoter was notcompletely suppressed in glycerol, and thus the background levels ofrecombination seen on glycerol may at least partially reflect this lackof complete suppression. To overcome this, strains were created whereinrdhA (PaHa32) or rdhB (PaHa33) was expressed under control of the weakerniaA promoter. The 3′-deleted copy of hemA carried on plasmid pPH7 wasintroduced into these strains in a manner identical to that describedabove for creation of PaHa31-2.2. The specific transformants selectedfor testing were designated Aspergillus oryzae PaHa32-4.6 andPaHa33-5.1.

Approximately 2×10⁷ spores of PaHa32-4.6 or PaHa33-5.1 were plated oneither MMASM (Example 15) or MMNSM (MMASM with 10 mM NaNO₃ in place ofNH₄Cl). The former medium keeps the niaA promoter turned off and thelatter medium induces the niaA promoter and hence stimulatestranscription of the rdhA or rdhB gene. The appearance of colonies wasmonitored for 7 days. The results demonstrated that interchromosomalrecombination is stimulated by an elevation in transcription of eitherrdhA or rdhB.

Example 18 PCR Amplification of a Portion of the Aspergillus oryzae rdhdGene

A portion of the Aspergillus oryzae rdhD (rad54 homolog D) gene wasamplified by nested degenerate PCR. The amplification employed primers980057, 980058, 980059 and 980060 shown below.

Primer 980057:

-   5′-GAYCCIGAYTGGAAYCCNG-3′ (SEQ ID NO:30)    Primer 980058:-   5′-TTYTTYTGICCRTCNCKCCA-3′ (SEQ ID NO:31)    Primer 980059:-   5′-AAYTAYACICARACNYTNGA-3′ (SEQ ID NO:32)    Primer 980060:-   5′-ATITTYTCYTCDATNGTNC-3′(SEQ ID NO:33)

The first amplification reaction (30 μl) was prepared using Aspergillusoryzae HB101 genomic DNA as template with the following components:PCRbuffer II (Perkin Elmer), 0.20 mM dNTPs, 0.4 μg of Aspergillus oryzaeHowB101 genomic DNA, 5.0 μM primer 980059, 5.0 μM primer 980060, and 3.0units of Taq DNA polymerase. Before amplification, the template DNA wasdenatured in a boiling water bath for 5 minutes and quick-cooled on ice.The reaction was initiated by adding Taq DNA polymerase to the otherreaction components at 72° C. The reactions were incubated in aStratagene Robocycler programmed as follows:35 cycles each for 45seconds at 94° C., 45 seconds at 39, 41, or 43° C., and 60 seconds at72° C. (7 minutes final extension). Reaction products were pooled,precipitated with 2 volumes of ethanol, dried, and dissolved in 10 μl ofTE. The second amplification reaction (30 μl) was prepared using theproduct of the first amplification as template with the followingcomponents:PCR buffer II (Perkin Elmer), 0.20 mM dNTPs, 0.2 μl oftemplate DNA, 5.0 μM primer 980057, 5.0 μM primer 980058, and 3.0 unitsof Taq DNA polymerase. Before amplification, the template DNA wasdenatured in a boiling water bath for 5 minutes and quick-cooled on ice.The reaction was initiated by adding Taq DNA polymerase to the otherreaction components at 72° C. The reactions were incubated in aStratagene Robocycler programmed as follows:35 cycles each for 45seconds at 94° C., 45 seconds at 46, 48, 50, or 52° C., and 60 secondsat 72° C. (7 minutes final extension).

A portion of the reaction products was fractionated on a 3% agarose gel,and bands at about 70 bp were excised and purified using QIAquick with afinal elution volume of 30 μl. Approximately 2 μl of this product wasreamplified under the same PCR conditions and fractionated and purifiedin the same manner. The ca. 70 bp fragment was cloned using the TOPO TAcloning kit according to the manufacturer's instructions and the DNAsequence was determined using T7 promoter primer. DNA sequence analysisof the 68 bp gene segment showed that the amplified gene encoded aportion of the Aspergillus oryzae rdhD gene. The sequence from thisclone was used to design a non-degenerate primer to be used foramplification of a larger region of the rdhD gene. The employed primeris shown below.

Primer 980866:

-   5′-AATGCTTGTTGATCAGCAG-3′ (SEQ ID NO:34)

The amplification reaction (120 μl) was prepared using Aspergillusoryzae HB425 genomic DNA as template with the following components:PCRbuffer II (Perkin Elmer), 0.25 mM dNTPs, 2.0 μg template DNA, 4.2 μMprimer 980059, 0.4 μM primer 980866, and 5.0 units of Taq DNApolymerase. Before amplification, the template DNA was denatured in aboiling water bath for 5 minutes and quick-cooled on ice. The reactionwas initiated by adding Taq DNA polymerase to the other reactioncomponents at 72° C. The reactions were incubated in a StratageneRobocycler programmed as follows:30 cycles each for 45 seconds at 94°C., 45 seconds at 39, 41, 43, or 45° C., and 60 seconds at 72° C. (7minutes final extension). The ca. 250 bp product was fractionated on anagarose gel, excised, and purified using the QIAquick system. Three μlof the purified fragment was reamplified under the same PCR conditionsfor 25 cycles at an annealing temperature of 40° C., and the product wasgel purified in the same manner. Direct sequencing of the PCR productusing primer 980866 demonstrated that the gene fragment encoded aportion of the rdhD gene.

Example 19 Isolation of Partial-length Aspergillus oryzae rdhD GenomicClones

Genomic libraries were prepared and plated as in Example 3. The PCRproduct of 232 bp described in Example 18 was radioactively labeledusing the Strategene Prime-It II kit according to the manufacturer'sprotocol with the exception that the random primers were replaced by 0.6μM of primer 866. The labeled product was used to probe approximately100,000 pfu of an Aspergillus oryzae genomic library constructed fromAspergillus oryzae strain HowB430 in the vector λZipLox using the sameprocedures described in Example 3.

Eleven hybridizing plaques were obtained, and four of these werepurified, excised as pZL1 clones, and characterized as described inExample 3.

Example 20 Characterization of the Aspergillus oryzae Genomic CloneEncoding RDHD

DNA sequencing of each clone was performed with an Applied BiosystemsPrism 377 DNA Sequencer using the BigDye Terminator Cycle SequencingReady Reaction kit according to the manufacturer's instructions.Oligonucleotide sequencing primers were designed to complementarysequences in the pZL1 plasmid vector and were synthesized by OperonTechnologies Inc., Alameda, Calif. Contig sequences were generated usinga transposon insertion strategy (Primer Island Transposition Kit,Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.).

A 5514 bp genomic fragment was sequenced to an average redundancy of6.0, and includes sequences from all of the genomic clones. No singleclone contained the entire gene, but overlapping pZL1 clones #10 and #17together encompassed the entire gene. The nucleotide sequence anddeduced amino acid sequence are shown in FIG. 2. Sequence analysis ofthe cloned insert revealed a coding sequence of 2645 bp (excluding thestop codon) encoding a protein of 811 amino acids. Clone 10 containednucleotides 390-2906 of SEQ ID NO:5 encoding amino acids 59-811 of SEQID NO:6, while clone 17 contained nucleotides 161-1749 of SEQ ID NO:5encoding amino acids 1-459 of SEQ ID NO:6. The coding sequence ispunctuated by four introns of 54 bp, 63 bp, 49, and 46 bp. The G+Ccontent of the coding sequence (including introns) is 47.3%. Thepredicted RDHD polypeptide has a molecular mass of 99.2 kDa and anisoelectric point of 8.90. Using the Signal P software program (Nielsenet al., 1997, Protein Engineering 10:1-6), no signal peptide waspredicted (Y<0.037).

A comparative alignment of the Aspergillus oryzae RDHD protein sequencewith other sequences using the Clustal W algorithm in the Megalignprogram of DNASTAR, showed that the deduced amino acid sequence of theAspergillus oryzae RDHD protein shares 74% identity with the deducedamino acid sequence of the MUS-25 protein of Neurospora crassa(accession number Q9P978).

Clones 10 and 17 were deposited as E. coli pZL1rdhD17 (NRRL B-30505) andE. coli pZL1rdhD10 (NRRL B-30506) on Jul. 27, 2001, with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession numbers:

Deposit Accession Number Date of Deposit E. coli pZL1rdhA13 NRRL B-30503Jul. 27, 2001 E. coli pZL1rdhB6  NRRL B-30504 Jul. 27, 2001 E. colipZL1rdhD17 NRRL B-30505 Jul. 27, 2001 E. coli pZL1rdhD10 NRRL B-30506Jul. 27, 2001

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent a substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method for increasing the homologousrecombination of a nucleic acid sequence in a population of filamentousfungal host cells, the method comprising: (a) introducing into thepopulation of the filamentous fungal host cells the nucleic acidsequence which comprises one or more regions which are homologous withone or more genomic regions of the filamentous fungal host cells andintroducing a second nucleic acid sequence comprising expression controlsequences directing expression of a nucleic acid sequence encoding afilamentous fungal recombination protein, wherein (i) the recombinationprotein promotes the recombination of the one or more regions with thecorresponding homologous regions in the genome of the filamentous fungalhost cell to incorporate the nucleic acid sequence therein by homologousrecombination, and (ii) the number of host cells comprising theincorporated nucleic acid sequence in the population of the filamentousfungal host cells is increased at least 20% compared to the samepopulation of filamentous fungal host cells without the second nucleicacid sequence; and (b) isolating from the population of the filamentousfungal host cells a filamentous fungal cell comprising the incorporatedsecond nucleic acid sequence; wherein the second nucleic acid sequenceencoding the recombination protein is selected from the group consistingof: (1) a nucleic acid sequence encoding a recombination proteincomprising or consisting of the amino acid sequence of SEQ ID NO: 4; and(2) a nucleic acid sequence comprising the nucleotide sequence of SEQ IDNO: 3, wherein the nucleic acid sequence encodes the recombinationprotein.
 2. The method of claim 1, wherein the recombination proteincomprises or consists of the amino acid sequence of SEQ ID NO:
 4. 3. Themethod of claim 1, wherein the first nucleic acid sequence furthercomprises (a) a gene that encodes a polypeptide or an RNA; (b) a genedisrupted with a third nucleic acid sequence; (c) a partially deletedgene; (d) a regulatory control sequence; or (e) a recombinant version ofa gene native or foreign to the filamentous fungal host cell.
 4. Amethod for producing a polypeptide in a filamentous fungal cell, themethod comprising: (a) cultivating the filamentous fungal cell in amedium suitable for production of the polypeptide, wherein thefilamentous fungal cell was obtained from a population of filamentousfungal cells produced by (a) introducing into the population of thefilamentous fungal host cell a first nucleic acid sequence comprisingexpression control sequences directing expression of a nucleic acidsequence encoding a filamentous fungal recombination protein and asecond nucleic acid sequence encoding the polypeptide or comprising aregulatory control sequence involved in the expression of a sequence inthe genome of the filamentous fungal cell encoding the polypeptide andfurther comprising one or more regions which are homologous with one ormore genomic regions of the filamentous fungal host cell, wherein (i)the recombination protein promotes the recombination of the one or moreregions with the corresponding homologous regions in the genome of thefilamentous fungal host cell to incorporate the second nucleic acidsequence therein by homologous recombination, and (ii) the number ofhost cells comprising the incorporated second nucleic acid sequence inthe population of the filamentous fungal host cells is increased atleast 20% compared to the same population of filamentous fungal hostcells without the first nucleic acid sequence; and (b) isolating fromthe population of filamentous fungal host cells a filamentous fungalcell comprising the incorporated first nucleic acid sequence; and (b)recovering the polypeptide from the cultivation medium; wherein thefirst nucleic acid sequence encoding the recombination protein isselected from the group consisting of: (1) a nucleic acid sequenceencoding a recombination protein comprising or consisting of the aminoacid sequence of SEQ ID NO: 4; and (2) a nucleic acid sequencecomprising the nucleotide sequence of SEQ ID NO: 3, wherein the nucleicacid sequence encodes the recombination protein.
 5. The method of claim4, wherein the recombination protein comprises or consists of the aminoacid sequence of SEQ ID NO:
 4. 6. The method of claim 4, wherein thesecond nucleic acid sequence further comprises (a) a gene, which encodesa polypeptide or an RNA; (b) a gene disrupted with a third nucleic acidsequence; (c) a partially or fully deleted gene; (d) a regulatorycontrol sequence; or (e) a recombinant version of a gene native orforeign to the filamentous fungal host cell.
 7. A method for deleting ordisrupting a gene in a filamentous fungal cell, the method comprising:(a) wherein the filamentous fungal cell is obtained from a population offilamentous fungal cells produced by introducing into the population ofthe filamentous fungal host cell a first nucleic acid sequencecomprising expression control sequences directing expression of anucleic acid sequence encoding a filamentous fungal recombinationprotein and a second nucleic acid sequence comprising one or moreregions which are homologous with one or more genomic regions of thefilamentous fungal host cell, wherein (i) the recombination proteinpromotes the recombination of the one or more regions with thecorresponding homologous regions in the genome of the filamentous fungalhost cell to incorporate the second nucleic acid sequence therein byhomologous recombination to delete or disrupt the gene in thefilamentous fungal cell, and (ii) the number of host cells comprisingthe incorporated second nucleic acid sequence in the population of thefilamentous fungal host cells is increased at least 20% compared to thesame population of filamentous fungal host cells without the firstnucleic acid; and (b) isolating from the population of filamentousfungal cells a filamentous fungal cell comprising the deleted ordisrupted gene; wherein the first nucleic acid sequence encoding therecombination protein is selected from the group consisting of: (1) anucleic acid sequence encoding a recombination protein comprising orconsisting of the amino acid sequence of SEQ ID NO: 4; and (2) a nucleicacid sequence comprising the nucleotide sequence of SEQ ID NO: 3,wherein the nucleic acid sequence encodes the recombination protein. 8.The method of claim 7, wherein the recombination protein comprises orconsists of the amino acid sequence of SEQ ID NO:
 4. 9. The method ofclaim 7, wherein the second nucleic acid sequence further comprises (a)a gene disrupted with a third nucleic acid sequence; (b) a partiallydeleted gene; (c) a DNA fragment; or (d) a selectable marker gene.